Drought, salinity, and extreme temperatures are the most common environmental stresses that adversely affect plant growth and development. These stresses limit plant productivity in cultivated areas worldwide. Under current, changing climate conditions, stress tolerance and crop yields under stress must be improved to supply food and fiber for an increasing world population.
Plants respond to abiotic stresses through various pathways. There is an unfilled need for improved stress tolerance in crops. Field crops, such as rice (Oryza sativa L.) or cotton (Gossypium hirsutum L.), typically experience more stress under similar conditions than do native plants growing wild in the same locale, for example native halophytes or native poikilohydric plants (“resurrection plants”). Rice plants, for example, are sensitive to salt, drought, and cold temperatures. Rice suffers from moderate to severe declines in both growth and productivity when exposed to drought or salt stress. Most rice plants grow poorly or not at all if the salt concentration is above ˜50 mM NaCl, whereas the halophyte Spartina alterniflora (Loisel), also known as smooth cordgrass, can complete its entire life cycle under salinity as high as 500 mM NaCl. While cotton is generally more drought-tolerant than rice, high and consistent cotton yields are better achieved with supplemental irrigation in times of drought. Avoiding stress is particularly important during critical reproductive phases, and is one reason why over one-third of US cotton acreage is under some form of irrigation. External irrigation is costly. Additionally, the quality of available water for irrigation has declined in many locations, and available water is often higher in dissolved salts, which places additional stress on plants. Cotton fiber quality can decline when the plants suffer from drought or salinity stress.
In contrast to glycophytes (plants that are not salt-tolerant, such as rice or cotton), halophytes (salt-tolerant plants) have adaptations at physiological, cellular, and molecular levels that help the plants cope with higher salt concentrations. These mechanisms include ion homeostasis, osmotic adjustment, ion extrusion, and compartmentalization. Spartina alterniflora is a perennial deciduous grass, a halophyte that is native to intertidal saline marshes along the Atlantic coasts of North and South America, and the Gulf of Mexico. S. alterniflora is a facultative halophyte that accumulates Na+, sequesters it in vacuoles, and excretes excess NaCl through specialized salt glands. S. alterniflora also synthesizes compatible solutes such as proline, glycine betaine; and it excludes ions from absorption by its roots. See Baisakh et al., Plant Science 170: 1141-1149 (2006). Identifying the genes responsible for stress tolerance in plants such as S. alterniflora, and transforming those genes into crop plants such as rice or cotton has the potential to create genetically modified crops with improved tolerance to abiotic stresses.
One way to improve the yields of cotton (or other crops) in drought and high salinity is to sequester cytosolic sodium into vacuoles, avoiding the accumulation of sodium ions at toxic levels in the cytoplasm, and achieving better water retention and higher salt tolerance. Vacuolar sodium sequestration is mediated by an active Na+/H+ “antiporter” membrane protein. The exchange of ions is driven by primary active H+ transport at the vacuole by V-ATPase. (V-ATPase is an ATP-dependent protein pump.) H+-ATPase acts as a primary transporter that pumps protons out of cytoplasm, creating pH and electric potential gradients across the vacuole membrane, thereby activating secondary transporters for ion and metabolite uptake. Baisakh et al., Plant Biotechnology Journal 10:453-464 (2012) reported that constitutive over-expression of a Spartina alterniflora gene (SaVHAc1) for c1 subunit vacuolar H+-ATPase gene conferred salt tolerance to transgenic rice plants.
Another approach to improving stress tolerance in field crops is to manipulate regulatory genes, such as those involved in signaling pathways, or transcription factors that modulate the downstream expression of stress-responsive genes. Park et al., BMC Plant Biology 12:90 (2012) recently identified a number of differentially-expressed mRNA transcripts in cotton plants subjected to water stress. Some of these transcripts were associated with heat shock and reactive oxygen species. Other researchers have identified dehydration-responsive element-binding genes, including DREB1 and DREB2. These genes are important in abscissic acid-independent stress tolerance pathways that interact with the cis-acting DRE (dehydration responsive element). Over-expression of the native form of DREB1 and of a constitutively-active form of DREB2 increases the tolerance of transgenic Arabidopsis plants to drought, salinity, and cold. Kasuga et al., Nature Biotechnology 17:287-291 (1999). Over-expression of DREB genes also increases tolerance of rice plants to salinity and drought. See Datta et al., Plant Biotechnology Journal 10:579-586 (2012).
The actin cytoskeleton is critical for many cellular processes, including several that are essential for plant development. These processes require constant reorganization and remodeling of the actin filament (F-actin) network. F-actin turnover involves polymerization, depolymerization, severing, nucleation, and large scale translocation events. The actin-binding proteins regulate the spatial configuration of actin arrays and dynamic cytoskeleton rearrangements. Actin-binding proteins sense environmental changes and influence actin filament polymerization, depolymerization, branching, and bundling.
Actin depolymerizing factor (ADF)/cofilins are a large family of ubiquitous, low molecular mass (15 to 20 kDa), actin-modulating proteins found in eukaryotic cells. As key regulators of the dynamics of actin arrays, these proteins play an important role in growth and development. ADF is phylogenetically conserved in plants, animals, and fungi. ADF specifically binds the actin-bound form of both monomeric (G-) and filamentous (F-) actin. ADF increases actin turnover by severing actin filaments, reducing filament length, and increasing barbed ends. ADF also increases dissociation of the F-actin monomer from the pointed ends by changing the helical twist of the actin filament, thereby accelerating the dissociation of actin subunits. Reversible phosphorylation, specific phosphoinositides, calcium-stimulated protein kinase, Rop GTPases, and pH all affect ADF activity in plants. ADFs also play a role in pollen tube growth, root formation, and cold acclimation.
Yan et al., Proteomics 5:235-244 (2005) reported a systematic proteomic investigations of salt stress-responsive proteins in rice. One of the proteins up-regulated following salt stress was identified as a putative actin-binding protein, that the authors suggested was probably a previously unreported ADF in rice.
Studying the regulation of plant ADF has been challenging because of the presence of numerous isoforms in higher plants. RNAi-mediated knockdown of ADF2 has been reported to interfere with cell growth and differentiation in Arabidopsis. Mass spectrometry showed up-regulation of ADF proteins in rice leaves after 23 days of water stress. See Salekdeh et al., Proteomics 2:1131-1145 (2002).
Ali and Komatsu, Journal of Proteomics Research 5:396-403 (2006) reported that ADF was up-regulated in rice leaf sheath after 2 to 6 days of drought stress. See also www.ncbi.nlm.nih.gov/nucest/EH277804.
Baisakh et al., Functional & Integrative Genomics 8:287-300 (2008) reported that an ADF-like protein was up-regulated in S. alterniflora under salt stress.
Ouellet et al., Plant Physiology 125:360-368 (2001) reported that ADF was up-regulated in wheat (Triticum aestivum) during cold stress.
Baisakh and Subudhi, Plant Biotech J. 47:232-235 (2009) reported down-regulation of ADF in leaves and a slight up-regulation in roots under heat stress in S. alterniflora 
Huang et al., The Rice J. 5:33: 1-35 (2012) reported that ADF3 was up-regulated in rice under drought stress. Overexpression of rice ADF3 was reported to confer drought tolerance to Arabidopsis. 
Increased expression of an Arabidopsis vacuolar pyrophosphatase gene, AVP1, was reported to enhance drought and salt tolerance in transgenic cotton. (Zhang et al., Plant Signaling & Behavior 6:861-863 (2011). The likely molecular mechanism of AVP1-mediated drought resistance was described as increased proton pump activity in vacuolar pyrophosphatase, which increases the proton electrochemical gradient across the vacuolar membrane. This gradient leads both to lower water potential in the plant vacuole and to higher secondary transporter activities, inhibiting toxic ion accumulation in the cytoplasm. Overexpression of AVP1 appeared to stimulate root development, and the larger root system allowed AVP1-overexpressing plants to absorb water more efficiently under drought and saline conditions, enhancing stress tolerance and increasing yields. Larger root systems or shifts in root/shoot ratio could improve cotton yields under water stress conditions.
Bedre et al., “Genome-wide transcriptome analysis of the halophyte grass Spartina alterniflora reveals molecular basis of its salt adaptation responses, Abstract and Poster, presentation # P795 at the Plant and Animal Genome XXII meeting, (Jan. 11-14, 2014, San Diego, Calif.) describes findings for leaf and root transcriptome analysis of Spartina alterniflora subjected to 500 mM NaCl.
Climate change can lead to unpredictable weather patterns, rises in sea level and saltwater incursions (salinity stress), erratic rainfall (water stress), and temperature fluctuations (cold and heat stress). These environmental stresses can adversely affect crop growth and productivity. There is an unfilled need for improved field crops that can better tolerate such abiotic stresses.